Extraction and identification of a plant toxic protein (abrin) by Liquid Chromatography Tandem Mass Spectrometry

The aim of this work was unequivocal identification of abrin that is a dangerous plant toxin, in 6 unknown samples that were sent for analysis. Abrin lethal dose is 0.1–1 μg/kg body weight so due to the potential misuse as a biothreat agent, abrin is in the focus of surveillance. Therefore a reliable and fast identification of toxin in potentially contaminated environmental or clinical samples at low level, is of great importance [1, 2]. The most reliable technique for unequivocal identification of abrin, is detection of its specific peptides after enzymatic digestion, using liquid chromatography tandem mass spectrometery technique and MRM method and also measuring the activity of sample to ensure that the protein isn’t denatured and acts properly in the body. Abrin is a 60 kDa, natural toxic protein isolated from beans of the tropical and subtropical leguminous plant Abrus precatorius. It has two A-chain and B-chain with a disulfide linked heterodimeric chains. The B-chain binds to cell surface receptors and facilitates a transport of the A-chain across the cell membrane. The A-chain is not active until it is internalized by the cell, where halts protein synthesis by removing an adenine within the α-sarcin site on the large (28S) ribosomal subunit, which results in failure of protein synthesis and death. There are some unique peptides after digestion in both chains that are specific for this protein and can be used for identifications [3]. In this work, after extraction of abrin from sample, enzymatic digestion (trypsin) and clean-up, 3 unique peptides from A-chain (T2A, T3A, T7A) and 2 unique peptides from B-chain (T3B and T4B), were identified using LC-MS/MS technique and MRM method in 2 contaminated samples with abrin concentration about 100 ppm, successfully. Also the activity assay was done by adding a RNA substrate (GCGCGAGAGCGC) to the sample and measuring the released adenine by LC-MS/MS and results showed that they were active.